The biotransformation of steroids by conjugation to a highly charged sulfonate group is an important process that leads to a marked change in the physicochemical properties of these essentially hydrophobic compounds. The sulfonation of steroids is brought about by the transfer of a sulfonate group (SO3-) to an appropriate hydroxyl acceptor site and is carried out by enzymes termed sulfotransferases. Steroid sulfonation, by altering molecular polarity, increases solubility in an aqueous milieu and modifies binding to protein; this in turn influences transportability and acts as an intracellular trapping or storage mechanism. Steroid sulfonation also impacts on biological responsivity by serving to either initiate an event (steroid sulfonate is the active form) or terminate an event (steroid sulfonate is the inactive form). Steroid sulfonation is particularly prominent in steroid-producing tissues; in fact, one of the most active tissues in this regard is the adrenal cortex where tissue-specific steroid sulfotransferases are differentially expressed in functionally distinct adrenocortical zones. The aim of the SSR program is to gain a fuller understanding of the precise biological consequences of steroid sulfonation. To achieve this goal the biochemical characterization and molecular biology of steroid-specific sulfotransferases, as well as ATP-sulfurylase and APS kinase, the catalytic activities responsible for production of the universal sulfonate donor (PAPS), have been undertaken. The cDNA for stereoselective 3beta-hydroxysteroid sulfotransferase (HST) has been cloned, expressed and found to be 87% identical to chiral-specific 3alpha-HST. The structural genes for estrogen sulfotransferase (EST) and 3beta-HST have been determined and the 5'-flanking regions cloned and analyzed; examination of transcriptional regulation is ongoing. An amino acid motif near the C-terminus, conserved in all steroid and phenol sulfotransferases, has been identified as the sulfonate donor binding site; furthermore, amino acid residues involved in estrogen interaction with EST have been specified by site-directed mutagenesis. Human PAPS synthetase, a single polypeptide chain containing both ATP-sulfurylase and APS kinase activities, has been cloned and expressed; additionally, constructs containing either the ATP-sulfurylase or APS kinase domains have been generated and expressed.